Technology & Science
We transform complex biology into precise, testable solutions—powered by validated siRNA and ASO technologies and decades of RNAi expertise.
Technology & Science
We transform complex biology into precise, testable solutions—powered by validated siRNA and ASO technologies and decades of RNAi expertise
The Science Behind siRNA and ASOs
The Science Behind
siRNA and ASOs
Oligonucleotide therapies such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are at the forefront of precision medicine. These engineered molecules provide powerful tools to silence, modify, or regulate gene expression — not by targeting proteins, but by acting upstream at the RNA level. Their unique mechanisms enable highly specific and tunable control over gene expression, and their modular chemistry allows for broad optimization across delivery strategies, biological half-life, tissue targeting, and immunogenicity.
How siRNA Works
siRNAs are typically 21–23 nucleotide-long double-stranded RNA molecules with 2-nucleotide 3’ overhangs. These molecules function by hijacking the endogenous RNA interference (RNAi) pathway, a conserved cellular mechanism that regulates gene expression and defends against viral genomes.
After entering a cell—via lipid-mediated transfection, electroporation, or chemical conjugates—the siRNA duplex is recognized by the RISC-loading complex. The guide strand is incorporated into the RNA-induced silencing complex (RISC), while the passenger strand is discarded.
The RISC complex uses the guide strand to scan intracellular mRNA transcripts for sequence complementarity. When a match is found, Argonaute-2 (AGO2), the catalytic core of RISC, cleaves the target mRNA at the center of the binding site. This cleavage triggers rapid degradation of the transcript and prevents protein translation.
Key Features of siRNA Activity:
- Requires near-perfect base pairing to the target
- Acts primarily in the cytoplasm
- Leads to irreversible degradation of the target mRNA
- Highly potent even at sub-nanomolar concentrations
- Effective in both dividing and non-dividing cells
How ASOs Work:
Antisense oligonucleotides (ASOs) are short, single-stranded DNA or RNA analogs typically 15–25 nucleotides in length. Unlike siRNAs, ASOs can modulate RNA function through multiple mechanisms depending on their structure and chemical composition.
RNase H-Mediated Cleavage:
Some ASOs bind to complementary mRNA sequences to form DNA–RNA hybrids that are recognized by RNase H1, which cleaves the RNA strand and facilitates degradation. These ASOs can act in both the nucleus and cytoplasm.
Steric Blocking:
Other ASOs work by simply binding to RNA and physically preventing interactions with proteins involved in splicing, translation, or other post-transcriptional processing. These ASOs do not cause degradation but alter transcript usage or translation efficiency.
Splice-Modulating ASOs:
A specialized subset of ASOs is designed to influence alternative splicing patterns by binding to pre-mRNA and promoting the inclusion or exclusion of specific exons. This approach is used in drugs like nusinersen (Spinraza) for spinal muscular atrophy. ASOs are often favored for targeting nuclear transcripts or achieving regulatory effects that do not require mRNA cleavage. Their single-stranded nature also makes them easier to synthesize and modify.
The Critical Role of Chemical Modifications
Unmodified oligonucleotides are vulnerable to degradation by nucleases and can inadvertently activate immune sensors like Toll-like receptors (TLRs). Chemical modifications are essential for improving the pharmacokinetics, potency, specificity, and safety of oligonucleotide drugs.
Common Modifications:
- Backbone modifications (e.g., phosphorothioates) improve nuclease resistance and increase plasma protein binding
- Sugar modifications (e.g., 2’-O-methyl, 2’-fluoro, and locked nucleic acids or LNAs) increase thermal stability and reduce off-target binding
- Base modifications (e.g., 5-methylcytosine, pseudouridine) can reduce immunogenicity or enhance RNA binding affinity
These chemical changes are strategically placed to retain the therapeutic effect while optimizing pharmacological performance. For example, siRNAs often contain fully modified passenger strands and unmodified or selectively modified guide strands to maintain RISC compatibility while improving durability.
Note: The examples listed above represent common modification strategies, but a wide range of other options exist and can be tailored to suit specific therapeutic goals.
Delivery by Design: Conjugates and Tissue Targeting
Delivering oligonucleotides to the correct tissue or cell type remains one of the greatest challenges in therapeutic development. Naked oligos have poor membrane permeability and often distribute nonspecifically. To improve biodistribution and cellular uptake, oligonucleotides are commonly conjugated to small molecules or ligands.
Common Conjugations:
- Cholesterol: Enhances membrane interaction and passive uptake
- Fatty acids like docosanoic acid (DCA): Improve plasma half-life and facilitate endocytosis
- GalNAc (N-acetylgalactosamine): Targets hepatocytes via the asialoglycoprotein receptor (ASGPR)
- Peptides or antibodies: Directs oligonucleotides to specific cell-surface receptors (e.g., transferrin receptor for CNS delivery)
The ideal conjugate depends on factors such as the target tissue, route of administration, and pharmacodynamic goals. Conjugates can greatly enhance tissue specificity, reduce the required dose, and extend duration of action.
Note: The list above includes representative examples, but many other conjugation strategies exist.
A Platform for Precision Medicine
Oligonucleotide therapeutics offer notable advantages:
- Ability to suppress or modulate genes with high specificity
- Reversible effects and tunable pharmacology
- Access to targets previously considered "undruggable" by other modalities
- Fast, programmable development cycles
- Broad tissue applicability—from CNS and liver to lung, eye, and skin
We apply the principles of RNA biology, chemical design, and translational science to guide clients from initial sequence design to optimized therapeutic leads. Whether validating an siRNA sequence, designing a conjugation strategy, or optimizing chemical modifications, our goal is to help realize the full potential of oligonucleotide-based therapies.
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Work directly with our experts to design, synthesize, and validate your siRNA and ASO therapeutic candidates.
Ready To Get Started?
Work directly with our experts to design, synthesize, and validate your siRNA and ASO therapeutic candidates.